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b. Set a reference point (any point in between your top and bottom)
c. Set the top by either engaging the stepper motor and focusing up or
entering in a number.
d. Set the bottom same as the top.
e. Set step size (the N button sets Nyquist-Shannon Sampling which is 2.7
times the theoretical resolution limit)
f. Now click the green check box in the top window so when you click single
it takes the whole z-stack (if you do this beforehand it will take a z-stack
while you are trying to set the z-stack up)
i. Always make sure that the stepper motor is engaged!
10. Zoom:
a. Click on the top right box with the cube in it
b. Use this corners of the box to zoom in and grab the middle to move it
around
c. To zoom out drag the field zoom bar in the XY window all the way to the
right
d. Check the XY tab to see what your pizel size is and whether you are
doing Nyquist-Shannon Sampling (2.7 times the resolution limit). You
know you have reached this because the note ‘not optimum pixel size’
disappears.
11. XY basic:
a. Pixel dwell time: this is how long the laser stays on one pixel. I usually
leave this at the minimum time.
b. Field zoom: the zooming that you have done in step 10.
c. Pixels: I recommend using either 512x512 or 1024x1024 but you can do
arbitrary if you want
12. Time:
a. Use this to set up your time-series: interval and number of acquisitions.
Notes:
1. Saving Files:
a. Always save them as .ids (this will save 2 files: a .ids and .ics and both of
them are needed to open it at a later date)
2. Spectral Un-mixing: (only for when using the spectral detector)
a. Draw a ROI on image on each single color that you want to unmix (just
red or just green – it can be useful to have samples that are only stained
with one color for this)
b. Menu: Spectral
Unmix
follow script.
3. 3 Dimensional Rendering:
a. Data
Volume
Volume Render
4. 3D Orthogonal Rendering: (my favorite way to image a z-stack in EZ-C1)
a. Go to view and then in the drop down menu select 3D Orthogonal
i. This allows you to image XY, XZ, and YZ at the same time
